Strobilurin X acts as an anticancer drug by inhibiting protein synthesis and suppressing mitochondrial respiratory chain activity

Purpose Strobilurins act as antifungal agents by inhibiting the mitochondrial respiratory chain. The cytotoxic activity of strobilurins, focusing on its anticancer activities, has been reported. However, the mechanisms involved in these activities remain unclear. Methods The cytotoxic effects of strobilurin X isolated from the mycelium of Mucidula. venosolamellata were examined in human cancer cell lines (A549 and HeLa) and normal fibroblasts (WI-38). Results Strobilurin X significantly decreased the viability of A549 and HeLa cells compared to that in the WI-38 cells after 48 h of exposure. The EC50 values for cytotoxicity in the A549, HeLa, and WI-38 cells were 3.4, 5.4, and 16.8 μg/mL, respectively. Strobilurin X inhibited the mitochondrial respiratory chain and enhanced the release of lactate in the A549 cells. The IC50 value of strobilurin X against the mitochondrial respiratory chain complex III activity was 139.8 ng/mL. The cytotoxicity induced by strobilurin X was not completely rescued after adding uridine, methyl pyruvate, or N-acetyl cysteine. Furthermore, pharmacological approaches demonstrated that strobilurin X failed to modulate the mitogen-activated protein kinase family and phosphoinositide 3-kinase-Akt pathways; alternatively, it suppressed protein synthesis independent of uridine. Conclusion Strobilurin X induced cytotoxicity by blocking the mitochondrial respiratory chain and suppressing protein synthesis. These findings may aid in the development of novel anticancer drugs using strobilurins.


Introduction
Strobilurins are antifungal pesticides widely used in agriculture across the world [1,2].Strobilurins A and B were initially isolated from the mycelium of the basidiomycete Strobilurus tenacellus [3].Mucidin extracted from Oudemansiella mucida [4] was structurally identical to strobilurin A [5]. Strobilurins exert antifungal effects by inhibiting the transfer of an electron in the mitochondrial respiratory chain at complex III [5][6][7][8][9].Several types of strobilurins have been isolated and identified from various mushrooms and synthesized for the development of pesticides [1,10].Strobilurin X was detected in the culture medium of Oudemansiella mucida and showed the higher antifugal activity than strobilurin A [11,12].
Cancer is the most common morbidity, leading to a growing demand for the development of novel drugs.Despite many studies on the anticancer effects of various mushroom extracts, the findings have not been exploited for clinical use [13].The mitochondrial respiratory chain is one of many therapeutic targets for cancer treatments because it is essential for cell proliferation [14].In one study, the deletion of complex III, one of the mitochondrial respiratory chain components, abolished tumor cell growth, resulting in the amplification of the tumor engrafted in mice [15].However, no compounds inhibiting complex III have been successfully used to develop anticancer drugs.Strobilurins inhibit the activity of complex III by suppressing cytochrome bc1 [5,13]; thus, they may be useful for the development of compounds that can inhibit cancer cell proliferation.
The aim of this study was to examine the cytotoxic effects of strobilurin X, isolated from Mucidula venosolamellata, on cancer cells.Strobilurin X significantly suppressed cell proliferation and inhibited the mitochondrial respiratory chain complex III activity.However, the cytotoxicity was not solely attributed to the suppression of complex III, indicating that strobilurin X had alternative targets for its cytotoxic effect.Further experiments revealed that strobilurin X inhibited protein synthesis.These results suggested that strobilurin X exerts anticancer effects and may serve as a lead compound for developing new anticancer drugs.

Cytotoxicity induced by strobilurin X
Strobilurins have been reported to induce cell death [19,20].Therefore, the cytotoxic effect of strobilurin X was examined in the current study.Strobilurin X significantly decreased the viability of the A549 and HeLa cells at 48 h in a concentration-dependent manner.The EC 50 values of strobilurin X in the A549 and HeLa cells were 3.4 ± 0.2 and 5.4 ± 0.3 μg/mL, respectively (Figs. 1A and B).Although a cytotoxic effect was observed in the WI-38 cells, the EC 50 value in these cells (16.8 ± 3.4 μg/mL) was higher than those in the two cancer cell lines (Fig. 1C).These results suggest that strobilurin X has a relatively selective cytotoxic effect on cancer cells.

Involvement of blockade of mitochondrial respiratory chain complex III in strobilurin X-induced cytotoxicity
A549 cells were used to evaluate the detail cytotoxic effects and mechanisms of strobilurin X. Inhibition of the mitochondrial respiratory chain enhances the glycolytic system [21]; therefore, the amount of lactate effluxed by glycolysis from the A549 cells was therefore measured in the current study.Strobilurin X increased lactate release at 48 h (Fig. 2A) and inhibited the activity of the mitochondrial respiratory chain complex III in a concentration-dependent manner.The IC 50 of strobilurin X was 139.8 ± 32.0 ng/mL; however, this value was significantly lower than the concentration showing cytotoxicity in A549 (Fig. 2B).These results clearly showed that strobilurin X inhibited mitochondrial respiratory chain complex III, although the experimental setting was different between the measurement of complex III activity and cytotoxicity.Future experiments using extracts from used cells may reveal details.
As shown in Fig. 3A, uridine (50 μg/mL) significantly reduced the cytotoxicity of strobilurin X, thus indicating that pyrimidine biosynthesis was inhibited by strobilurin X through the mitochondrial respiratory chain.Uridine rescued the cytotoxicity of strobilurin X (6.25 μg/mL) in a concentration-dependent manner, but did not completely overcome the cytotoxic effect even at 200 μg/mL (Fig. 3B).Pyruvate is also known to reduce the cytotoxicity induced by mitochondrial respiratory chain dysfunction [15,22].As shown in Fig. 3C, methyl pyruvate did not attenuate the strobilurin X-induced cytotoxic effect.These results suggest that other targets, besides the inhibition of the mitochondrial respiratory chain complex III, may be involved in the cytotoxic action of strobilurin X.

Inhibition of protein synthesis by strobilurin X
Among the cell growth signals, PD98059, U0126, mitogen activated protein kinase (MAPK)/extracellular signalregulated kinase (Erk) kinase (MEK) inhibitors, and LY2940001, a phosphoinositide 3-kinase (PI3K) inhibitor, failed to modulate the strobilurin X (6.25 μg/mL)-induced cytotoxicity.Likewise, among the cell death signals, JNK inhibitor II and SB203580, a p38 MAPK inhibitor, did not affect the strobilurin X-induced cell death (Fig. 4).These results suggest that the cell growth and cell death signals are not related to the cytotoxic effect of strobilurin X.
Various anticancer compounds have generated intracellular ROS, which induced cellular damage [23,24].The number of A549 cells emitting stronger fluorescence for the ROS indicator dichlorodihydrofluorescein (DCF) increased to the maximum level after 12 h of exposure to strobilurin X, although the mean fluorescence intensity was lower than that of number of hydrogen peroxide (0.5 mM) for 1 h (Fig. 5A and B).The strobilurin X-induced high fluorescent intensity levels were maintained up to 24 h (Fig. 5B).However, N-acetyl cysteine, a potent ROS scavenger, failed to attenuate the strobilurin X-induced cytotoxicity in A549 cells (Fig. 5C).These results suggest that strobilurin X induces the production of intracellular ROS, but its effect is not essential for the cytotoxicity.
Recently, the link between protein synthesis inhibition and anticancer activity has been focused on certain compounds [25].Protein synthesis is indispensable in cell proliferation; hence, we evaluated the protein synthesis activity using OPP, which was labeled on the C-terminus of nascent polypeptide chains [26].Strobilurin X decreased the cell fluorescent intensity labeled by protein synthesis for 2 h in A549 cells, indicating that strobilurin X inhibits protein synthesis (Fig. 6A).Uridine failed to block the inhibition of protein synthesis by strobilurin X (Fig. 6B).These results suggest that strobilurin X can inhibit protein synthesis and mitochondrial respiratory chain complex III.

Discussion
In this study, we found that the cytotoxic effects of strobilurin X (isolated from M. venosolamellata) on cancer cells were greater than those on normal fibroblasts.Moreover, strobilurin X inhibited protein synthesis and mitochondrial respiratory chain complex III in the lung cancer cell line.
The mitochondrial respiratory chain is essential for generating the intracellular concentration of ATP.It is considered as a target for anticancer effects owing to its ability to regulate the metabolism and proliferation of cancer cells [27].The oxidation of dihydroorotate dehydrogenase by complex III produces orotates, which lead to pyrimidine biosynthesis [28].The depletion of complex III inhibits de novo pyrimidine synthesis and induces cytotoxicity in cancer cells [15].A commercially available strobilurin pesticide, azoxystrobin, was reported to induce cytotoxicity, which was entirely rescued by uridine in myelogenous leukemia HL-60RG cells [29].In the current study, strobilurin  5 ROS production is not involved in the cytotoxicity induced by strobilurin X.A Typical histograms showing the cell numbers with DCF fluorescence after exposure to vehicle (12 h), strobilurin X (6.25 μg/mL, 12 h) and hydrogen peroxide (H 2 O 2 , 0.5 mM, 1 h).The mean fluorescence intensity (MFI) was calculated using the BD FACSDiva ™ software.B The time course of ROS production, indicated as the ratio of each MFI to that of the vehicle at each time point after exposure to strobilurin X. Symbols with vertical lines show the mean ± SEM in three separate experiments.The dashed line shows a baseline equal to the level in the vehicle.*p < 0.05 vs. the vehicle using Student's t-test.C The effect of N-acetyl cysteine (NAC; 1 mM) on strobilurin X-induced cytotoxicity in A549 cells at 48 h.Cell viability is indicated as the ratio of each value to that of the vehicle without NAC (open columns, vehicle; closed columns, strobilurin X [6.25 μg/mL]).Cells were pretreated with NAC 30 min before exposure to strobilurin X. Columns with vertical lines show the mean ± SEM in three separate experiments X-induced cytotoxicity was partially abolished by uridine, whereas methyl pyruvate did not have any effect in attenuating the strobilurin X-induced cytotoxicity (Fig. 3).Therefore, we hypothesized that other mechanisms, apart from mitochondrial respiratory complex III inhibition, were involved in the cytotoxicity of A549 cells.
Strobilurin X was found to directly suppress protein synthesis in this study (Fig. 6).Strobilurins A and B were reported to attenuate the incorporation of radioactive leucine in Ehrlich carcinoma ascites cells [3].Direct evidence for the inhibition of protein synthesis by strobilurin X was seen for the first time in the present study.The chemical structure of strobilurin X is different from that of strobilurin A in terms of the presence of a methoxy group at the para position of its benzene ring; it is different from that of strobilurin B at the position of the methoxy group and the Cl substituents and significantly different from that of azoxystrobin [1,3,11].Therefore, the inhibition of protein synthesis may be dependent on the structure of the strobilurins.Nonetheless, future studies identifying the molecular targets and clarifying the structure-activity relationship in protein synthesis are warranted.
Strobilurins are generally used as pesticides but are currently being evaluated as anticancer drugs [30].Interestingly, azoxystrobin induced cytotoxicity by inhibiting the phosphorylation of PI3K/Akt and Erk [31].However, the inhibitors of these molecules did not affect the cytotoxic action of strobilurin X in the present study.The cytotoxic activity of strobilurin X may not be related to these cell growth signals in A549 cells.
Natural compounds such as phytochemicals often generate ROS, which evokes cytotoxicity [23,24].Strobilurin X induced ROS generation in the current study.It may induce the generation of ROS in mitochondria due to the inhibition of mitochondrial respiratory chain complex III [32].However, the potent antioxidant N-acetyl cysteine failed to https://doi.org/10.1007/s12672-024-01041-wsuppress the strobilurin X-induced cytotoxicity in this study (Fig. 5).Similarly, mito-TEMPO, an intra-mitochondria ROS scavenger, also failed to block the strobilurin X-induced cytotoxicity (data not shown).Furthermore, strobilurin X did not induce the generation of ROS in HeLa cells despite its cytotoxicity (Fig. 1 and data not shown).Therefore, the generation of ROS by strobilurin X may not be essential for its cytotoxic action.Alternatively, the amount of ROS generated by strobilurin X may not be sufficient to kill A549 cells.
In summary, the inhibition of mitochondrial respiratory chain complex III and protein synthesis was found to be related to the cytotoxic effects of strobilurin X in A549 cells.Strobilurin-related compounds are widely used as pesticides; thus, the drug repositioning may be expected.The findings of this study indicate that strobilurin X may be considered for the development of novel anticancer drugs.

Fig. 1
Fig.1Cytotoxicity induced by strobilurin X.A Chemical structure of strobilurin X. Relationships between viability and the concentrations of strobilurin X at 48 h in the A549 B, HeLa C, and WI-38 D cells.Cell viability is indicated as the ratio of each value to that of the vehicle.Symbols with vertical lines show the mean ± the standard error of the mean (SEM) in three separate experiments

Fig. 3
Fig.3 Effects of uridine on the strobilurin X-induced cytotoxicity in A549 cells.A Relationships between the cell viability and concentrations of strobilurin X at 48 h without (•) and with uridine (○; 50 μg/ mL).Cell viability is indicated as the ratio of each value to that of the vehicle without uridine.Symbols  with vertical lines show the mean ± SEM in three separate experiments.B, C Relationship between the cell viability induced by the vehicle (open columns) and strobilurin X (closed columns, 6.25 μg/mL) and the concentrations of uridine B or methyl pyruvate (C; 110 μg/mL) at 48 h.Columns with vertical lines show the mean ± SEM in three separate experiments.*p < 0.05 vs. the vehicle with uridine using Student's t-test or Welch's t-test

Fig. 4
Fig. 4 Effects of inhibitors of cell proliferation and death signals on the cytotoxicity induced by strobilurin X in the A549 cells.Cell viability is indicated as the ratio of each value to that of the vehicle without inhibitors at 24 h (open columns, vehicle; closed columns, strobilurin X [6.25 μg/mL]).Veh, vehicle; PD, 10 μM PD98059; U01, 10 μM U0126; LY, 5 μM LY2940004; JNKi, 5 μM JNK inhibitor; SB, 10 μM SB203950.Columns with vertical lines show the mean ± SEM in three separate experiments

Fig. 6
Fig. 6 Effect of strobilurin X on protein synthesis in A549 cells.A Typical fluorescent microscope images of the protein synthesis activity 2 h after exposure to strobilurin X (upper, vehicle; lower, 25 μg/ mL of strobilurin X). White scale bars: 50 μm.B The fluorescent amount in each well without (vehicle) or with uridine (50 μg/mL) (open columns, vehicle; closed columns, 25 μg/mL of strobilurin X).Columns with vertical lines show the mean ± SEM in three separate experiments.*p < 0.05 vs. the vehicle using the Games-Hawel test.NS not significant